ANALYTICAL METHOD AND IMMUNOLOGICAL TREATMENT FOR BLADDER CANCER
20220265799 · 2022-08-25
Inventors
- Falk Büttner (Hannover, DE)
- Charlotte Rossdam (Hannover, DE)
- Rita Gerardy-Schahn (Hannover, DE)
- Astrid Oberbeck (Hannover, DE)
- Hossein Tezval (Hannover, DE)
Cpc classification
C07K2319/33
CHEMISTRY; METALLURGY
International classification
Abstract
The invention provides an analytical method for detecting bladder cancer by analysing a urine sample for presence of a glycan tumour antigen, the presence of which has been identified to indicate bladder cancer. The analytical method has the advantage that the tumour antigen can be analysed in a urine sample and has a high accuracy for indicating presence of cancer in the urinary tract, especially bladder cancer, e.g. the method has a low rate of false positive results.
Claims
1. A method for analysing a sample to detect cancer in the urinary tract and/or in the urogenital tract, the method comprising analysing a urine sample for the presence of the glycan consisting of Galβ1-4GlcNAcβ1-3Galβ1-4Glc (lacto-N-neo-tetraose, neolactotetra, nLc4), comparing a concentration of one or more of nLc4, lactose, and Gb3 to a control group and identifying the sample as indicting presence of cancer when the concentration is high compared to the control group and indicating lack of cancer when the concentration is low compared to the control group.
2. The method according to claim 1, wherein the analysing comprises contacting the sample with a peptide having specificity for binding nLc4.
3. The method according to claim 1, wherein the analysing comprises capillary gel electrophoresis of glycans obtained by hydrolysis of glycosphingolipids of a cell-free membrane vesicle fraction isolated from the urine sample.
4. The method according claim 1, wherein the identifying comprises determining a molar concentration of at least 1-fold of the internal standard Oligomannose 6 (Man6) or of at least 1% of nLc4 of the total of glycans originating from glycosphingolipids of the sample indicates presence of cancer in the urinary tract and/or in the urogenital tract.
5. The method according to claim 4, wherein the molar concentration is determined as the signal intensity.
6. The method according to claim 1, comprising additionally analysing the urine sample for presence of the glycan consisting of lactose.
7. The method according to claim 1, comprising additionally analysing the urine sample for presence of the glycan consisting of GalNAcβ1-3Galα1-4Galβ1-4Glc (globotetraose, Gb4).
8. The method according to claim 1, wherein the cancer is cancer of the bladder, the renal pelvis, the ureter or the urethra, e.g. urothelial cell carcinomas, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, sarcoma, paraganglioma, melanoma or lymphoma, or endometrial cancer.
9. The method according to claim 1, wherein the analysing comprises preparing a membrane fraction from the urine sample, then preparing a lipid fraction, and analysing the lipid fraction for the presence of the glycan.
10. (canceled)
11. A peptide, which is an antibody or a CAR, the peptide having specificity for binding a glycan consisting of nLc4, for use in the treatment of cancer in the urinary tract.
12. The peptide according to claim 12, wherein the peptide is a portion of an antibody or of a CAR that specifically binds to nLc4.
13. The peptide according to claim 12, wherein the peptide is a CAR expressed in a T-cell.
14. The peptide according to claim 12, wherein the cancer in the urinary tract and/or in the urogenital tract is a cancer of the bladder, of the renal pelvis, of the ureter or of the urethra, or endometrial cancer.
15. A compound comprising or consisting of nLc4 and/or nLc4-ceramide and/or a nLc4-protein conjugate and/or a nLc4-derivative for use in the treatment of cancer in the urinary tract and/or in the urogenital tract.
16. The compound according to claim 16, in a formulation suitable for i.m. or for systemic administration.
17. The compound according to claim 16, for use as a vaccine in the treatment or prevention of cancer in the urinary tract.
18. The compound according to claim 16, wherein the cancer is a cancer of the bladder, of the renal pelvis, of the ureter or of the urethra, or endometrial cancer.
19. The method according to claim 1, comprising additionally analysing the urine sample for presence of the glycan consisting of Galα1-4Galβ1-4Glc (globotriaose, Gb3).
Description
[0029] The invention is now described by way of examples and with reference to the figures, which show in
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EXAMPLE 1: DETECTION OF BLADDER CANCER BY XCGE-LIF ANALYSIS OF GLYCANS IN URINE SAMPLES
[0037] As an example for an analytical method, xCGE-LIF was used for the analysis of glycans that were obtained by hydrolysis of glycosphingolipids by ceramide glycanase of the membrane-vesicle fraction obtained from the cell-free liquid fraction of urine samples. In detail, urine samples, e.g. 40 mL, were centrifuged at 200×g for 20 min at 4° C., and the supernatant was centrifuged at 2000×g for 20 min at 4° C. to prepare a liquid cell-free fraction. This cell-free fraction was ultra-centrifuged in a Beckman-Coulter Optima L-100K centrifuge at 12500 rpm (16039×g) for 20 min at 4° C. and filtrated through a 0.22 μm membrane filter for removing residual cell debris. The liquid cell-free urine fraction was then ultra-centrifuged in a Beckman-Coulter Optima L-100K centrifuge using a Type 70 Ti rotor at 34200 rpm (120066×g) for 70 min at 4° C. to pellet the membrane-vesicle fraction. The supernatant was removed, and the pellet containing the membrane vesicles was washed by resuspending in 5 mL PBS (phosphate buffered saline) and again ultra-centrifuged using a Type 70 Ti rotor at 34200 rpm (120066×g) for 70 min at 4° C. to pellet the membrane vesicle fraction again.
[0038] The pellet was resuspended in 1 mL 1:2 v/v chloroform/methanol, preferably by firstly adding the methanol portion and then adding the chloroform portion. Solubilisation was preferably supported by exposition to ultrasound. The solubilized chloroform/methanol mixture was centrifuged at 1620×g for 5 min, and the supernatant was collected. The extraction of glycosphingolipids from the pellet was repeated twice using chloroform/methanol (2:1 (v/v)) and chloroform/methanol (1:1 (v/v)), respectively and the supernatants of the different extraction steps were pooled. The pooled supernatants were evaporated to dryness be removing the solvent, e.g. by a nitrogen stream. The dried glycolipids were dissolved in 1 mL methanol plus 2 mL water and then desalted applying on a Chromabond® C.sub.18 ec polypropylene column that was pre-equilibrated with a sequence of chloroform, then 1:1 v/v chloroform/methanol, then methanol, then water. After applying the solubilized glycolipids to the pre-equilibrated column, the column was washed 3× with water, then the glycolipids were eluted by methanol. The eluted glycolipids were again evaporated to dryness under nitrogen gas and stored at −20° C. until further use.
[0039] The eluted glycolipids were hydrolysed by ceramide glycanase derived from Hirudo medicinalis (GCase, obtained from Ludger, Oxfordshire, GB) in LudgerZyme ceramide glycanase reaction buffer for 24 h at 37° C., or with endoglycoceramidase I (EGCase I, obtained from NewEngland Biolabs, Ipswich, USA) from Rhodococcus triatomea in the reaction buffer provided at 37° C. for 16 h. These enzymes cleave the β-glycosidic bond of glycosphingolipids and release the free glycan with a reducing end. For xCGE-LIF, the free glycans were fluorescence-labelled by APTS, obtained from Merck, Darmstadt, Germany.
[0040] The glycans were mixed with 2 μL 20 mM APTS in 3.5 M citric acid, 2 μL 2-picoline borane complex (2 M in DMSO, obtained from Merck) and 2 μl water and incubated for 16.5 h at 37° C. in the dark. The reaction was stopped by adding 100 μL acetonitrile/water (80:20). Excess reagents were removed by hydrophilic liquid interaction chromatography applying Biogel P-10 gel (BioRad, Hercules, USA) in water/ethanol/acetonitrile (70:20:10) applied to the GHP membrane of a Pall Nanosep MF Centrifugal device (0.45 μm pore size, obtained from Merck). For pre-equilibration of the gel, solvent was removed by centrifugation at 54×g for 1 min, and the gel was washed 3× with water and then equilibrated with acetonitrile. Samples were applied to the suspended gel in 100 μL acetonitrile/water (80:20) with agitation for 5 min, liquid was removed by centrifugation at 54×g for 1 min, discarding flow through, washing the gel 5× with acetonitrile/water (80:20) containing 100 mM triethylamine (pH 8.5), then 3× acetonitrile/water (80:20), wherein in each washing step the gel was shaken for 1 min at 450 rpm and centrifuged at 54×g for 1 min. Elution of labelled glycans was done for three times with water by centrifugation of the gel upon incubation for 5 min at 450 rpm by shaking, respectively. The pooled eluates were then concentrated (SpeedVac) by evaporating the water in the dark and optionally stored at −20° C.
[0041] For xGCE-LIF, the resulting APTS-labelled glycans were suspended in water, e.g. 30 LC-MS grade (Merck), and 3 μl of glycans were mixed with 1 μL GeneScan 500 LIZ dye size standard (DNA fragments of 20 to 500 bp for use in calibration of MTU, 1:50 dilution in Hi-Di Formamide (obtained from Applied Biosystems, Thermo Fisher Scientific, Foster City, Calif.)) and added up to 10 μL with Hi-Di formamide in a MicoAmp Optical 96-well reaction plate (obtained from Applied Biosystems, Thermo Fisher Scientific, Foster City, Calif., USA). The mixture of ATPS-labelled glycans and the LIZ-labelled size standard was separated by multiplexed capillary gel electrophoresis (xCGE) on an ABI PRISM 3100-Avant Genetic Analyzer, remodeled by Axel Kunzler, advanced biolab service GmbH, Munich, Germany.
[0042] The analyser was equipped with an array of 4 capillaries of 50 cm length that were filled with POP-7 polymer (obtainable from Applied Biosystems, Thermo Fisher Scientific). Running buffer was obtained from Applied Biosystems, Thermo Fisher Scientific. Samples were injected for 15 sat 1.6 kV, and after a data delay of 50 s, detection was for 30 min at 12 kV at 60° C. using laser induced fluorescence detection and for analysis, the GeneMapper software v. 3.7 was used. For each measurement, an electropherogram was obtained displaying peak values for the size standard in one channel and for APTS-labelled glycans in another channel at specific migration time units (MTU) and with specific intensities (relative fluorescence units, RFU). A peak amplitude threshold of at least 10 RFU was set in the GeneMapper software for defining peaks.
[0043] For identification, known glycans were labelled with APTS and their MTUs were measured. Known glycans were used directly or after exoglycosidase digest using known enzymes. The following glycans were obtained and used as standards:
TABLE-US-00001 product name structure supplier name of glycan Glyko ® Lacto-N-Fucopentaose III Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc Prozyme Lewis.sup.x pentaose (LNFPIII) Glyko ® Lacto-N-Fucopentaose I Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc Prozyme fucosyl Lc4/SSEA5 (LNFPI) Blood group H antigen pentaose type Fucα1-2Galβ1-4GlcNAcβ1-3Galβ1-4Glc Elicityl-OligoTech ® fucosyl nLc4 2/Lacto-N-neofucopentaose I (LnNFPI) LS-Tetrasaccharide a (LSTa) Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc Elicityl-OligoTech ® sialyl Lc4 SSEA-4 hexaose Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galα1-4Galβ1-4Glc Elicityl-OligoTech ® sialyl Gb5/SSEA4 Globo H Fucα1-2Galβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glc Elicityl-OligoTech ® turesyl Gb5/globoH Globotriaose (Gb3) Galα1-4Galβ1-4Glc Elicityl-OligoTech ® Gb3 Lacto-N-neotetraose (LNnT) Galβ1-4GlcNAcβ1-3Galβ1-4Glc Elicityl-OligoTech ® nLc4 Globopentaose (Gb5) Galβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glc Elicityl-OligoTech ® Gb5/SSEA3 Blood group A antigen hexaose type 1 GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-3Galβ1-4Glc Elicityl-OligoTech ® A type 1 hexa
[0044] Further, the following glycosphingolipids (GSL) were obtained and, 50 ng each, were hydrolysed and APTS-labelled as described above and used as standards:
TABLE-US-00002 product name structure supplier trivial name Ganglioside GM3 Neu5Acα2-3Galβ1-4Glcβ1Cer Avanti ® GM3 Disialganglioside GD.sub.1a Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1Cer Merck GD1a Ganglioside GD.sub.3 Neu5Acα2-8Neu5Acα2-3Galβ1-4Glcβ1Cer Merck GD3 Ganglioside GD.sub.1b Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glcβ1Cer Merck GD1b Ganglioside GD.sub.2 GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glcβ1Cer Merck GD2 GM1 glycolipid Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1Cer Ludger GM1
[0045] Avanti® is available at Polar Lipids Inc.
[0046] As further standards, the known glycans, subsequent to being labelled with APTS, were digested with at least one of the following exoglycosidases:
TABLE-US-00003 incubation enzyme supplier dilution time α(2-3,6,8)-Neuraminidase EY labs 1:10 1 h (250 mU/ml) β(1-3)-Galactosidase New England 1:10 3 h (10,000 U/ml) Biolabs ® β(1-4,6)-Galactosidase Prozyme 3:10 over night (25 U/ml) α(1-3,4,6)-Galactosidase New England 1:10 3 h (8,000 U/ml) Biolabs ® α(1-2,3,4,6)-Fucosidase Prozyme 4:10 24 h (2 U/ml) β-N-Acetylhexosaminidase New England 3:10 over night (5,000 U/ml) Biolabs ® β-N-Acetylglucosaminidase New England 3:10 over night (4,000 U/ml) Biolabs ® α-N-Acetylgalactosaminidase New England 1:10 over night (20,000 U/ml) Biolabs ®
[0047] Non-specific activity of the exoglycosidases could be excluded by incubation with non-target glycans and subsequent analysis by xCGE-LIF.
[0048] The migration times ranging from 15 to 350 MTU measured could be analysed using CorelDRAW 2017. The intensity of the highest peak in each electropherogram was set to 1 for normalizing the relative fluorescence units. For quantitative analysis of peak intensities, the relative signal intensity of each peak was determined. Therefore, the heights of all peaks in the range from 15 to 300 MTU were summed up and the height of individual peaks was calculated as a percentage of the summed-up peak heights. For statistical analysis, the GraphPad Prism 4 software v. 4 was used. In figures, p-values of <0.05 upon unpaired Student's t-test were regarded as statistically significant and are depicted or highlighted by asterisks.
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[0055] Table 1 shows individual signal intensities of the glycosphingolipid-derived glycans nLc4, lactose, Gb4 and Gb3 for all 60 persons analysed relative to the internal standard Man6. Based on the common medical examination, persons were grouped in the groups “bladder cancer” (13 persons, #1 to #13) or “control” (47 persons, #14 to #60) with the latter not suffering from bladder cancer. Based on calculation of sensitivity and specificity for nLc4 and lactose, cut-off values were defined (see Table 2) and values above the cut-off were highlighted in bold in this table. No signal for nLc4 was detected in the bladder cancer patients #10-#13. However two of these patients display high values for the other positive marker lactose. It is known that cancers are genetically heterogeneous and it deserves further investigation of whether some types of bladder cancer do not produce nLc4 or whether the absence of nLc4 was caused by erroneously retrieval or storage of urine in the clinic.
[0056] Generally preferably, midstream morning urine is taken and directly frozen. Generally preferably, the concentration of the urine has to be determined by analysis of e.g. creatinine and the here postulated glycan markers should be related to the urine concentration. In addition to bladder cancer, other types of cancer associated with the urinary tract and/or in the urogenital tract, e.g. renal transitional cell carcinoma, malignant melanoma of the bladder or endometrial cancer showed values for nLc4 and/or lactose above the threshold. Cancers like prostate carcinoma or renal cell carcinoma mostly did not show apparent values for the analysed glycans.
TABLE-US-00004 TABLE 1 Signal intensity relative to Man6 Diagnosis of other # Group nLc4 lactose Gb4 Gb3 types of cancer 1 bladder cancer 51.22 519.28 49.47 425.72 2 bladder cancer 14.36 137.62 14.32 42.23 3 bladder cancer 8.08 202.37 22.55 72.45 4 bladder cancer 5.31 280.88 28.19 152.75 renal transitional cell carcinoma 5 bladder cancer 4.61 210.61 122.39 317.39 6 bladder cancer 4.50 49.84 12.48 38.77 7 bladder cancer 4.50 52.49 5.77 22.11 8 bladder cancer 4.42 14.50 5.85 14.96 renal pelvis cancer 9 bladder cancer 1.76 150.12 23.53 58.76 10 bladder cancer 0.00 50.95 27.24 79.95 11 bladder cancer 0.00 43.00 20.60 59.60 12 bladder cancer 0.00 584.74 13.53 21.72 13 bladder cancer 0.00 380.50 12.10 17.10 14 control 0.00 14.87 6.84 21.26 prostate carcinoma 15 control 0.00 9.54 3.18 8.72 16 control 0.00 12.65 5.88 20.60 17 control 0.00 33.46 13.39 32.86 18 control 0.00 91.91 6.70 9.00 gynaecological cancer 19 control 0.00 17.42 7.71 19.50 20 control 0.00 7.59 3.41 4.17 prostate carcinoma 21 control 0.00 33.31 13.69 27.69 prostate carcinoma 22 control 0.00 14.31 10.56 12.38 23 control 0.00 457.50 27.38 28.44 renal transitional cell carcinoma 24 control 0.00 382.55 8.36 15.50 25 control 0.00 440.00 27.45 45.78 26 control 0.00 457.82 2.67 3.56 27 control 0.00 225.05 5.60 13.41 28 control 0.00 136.08 10.89 17.89 renal cell carcinoma 29 control 0.00 388.45 15.91 34.82 prostate carcinoma 30 control 0.00 210.33 9.81 27.10 31 control 0.00 181.73 14.36 37.59 32 control 0.00 62.26 3.85 21.29 33 control 0.00 42.67 18.11 57.83 34 control 0.00 56.06 12.06 28.24 35 control 0.00 48.54 13.81 35.23 renal cell carcinoma 36 control 0.00 41.25 4.55 14.15 37 control 0.00 12.39 7.96 28.98 prostate carcinoma 38 control 0.28 32.03 8.87 13.75 39 control 0.34 57.35 36.34 61.00 40 control 0.48 23.55 31.67 59.36 41 control 0.60 21.47 20.01 37.78 42 control 1.21 28.95 10.11 20.95 43 control 1.29 21.88 7.88 39.29 44 control 1.30 36.70 39.80 63.70 45 control 1.31 74.27 2.85 17.92 46 control 1.39 60.44 34.99 92.14 47 control 1.52 69.09 19.78 75.26 48 control 1.65 62.00 18.29 65.65 renal cell carcinoma 49 control 1.69 59.78 49.08 153.57 50 control 2.27 98.33 34.77 100.23 51 control 2.79 78.55 51.07 149.54 52 control 3.48 204.38 51.24 138.10 53 control 4.10 64.70 43.70 102.60 54 control 4.97 92.45 44.03 138.00 malignant melanoma grown into bladder 55 control 6.22 134.04 30.22 68.30 56 control 7.65 239.59 9.18 27.00 57 control 10.54 163.82 29.96 146.86 58 control 11.42 99.21 58.55 144.58 59 control 12.28 188.41 12.62 44.28 endometrial cancer 60 control 28.00 319.76 91.08 204.32
[0057] Table 2 shows determination of sensitivity and specificity for glycan intensities relative to the internal standard Man6 from urine samples of 60 persons of which 13 were diagnosed for bladder cancer and 47 did not suffer from bladder cancer. For the glycosphingolipid-derived glycan nLc4, a sensitivity of 62% and a specificity of 85% was obtained at a cut-off value of ≥4.42. For the glycosphingolipid-derived glycan lactose, a sensitivity of 62% and a specificity of 72% was obtained at a cut-off value of ≥137.62. A combined analysis of nLc4 and lactose led to the diagnosis “no bladder cancer” when both markers were below the above defined cut-off and led to the diagnosis “bladder cancer” if one of both markers was above the above defined cut-offs with a sensitivity of 85% and a specificity of 66%.
TABLE-US-00005 TABLE 2 Intensity Diagnosed Bladder Marker (cut-off) as cancer Control Sensitivity Specificity nLc4 <4.42 Negative 5 40 0.62 0.85 ≥4.42 Positive 8 7 lactose <137.62 Negative 5 34 0.62 0.72 ≥137.62 Positive 8 13 combined both values below Negative 2 31 0.85 0.66 analysis of cut-off nLc4 and at least one value Positive 11 16 lactose above cut-off
[0058] Table 3 shows statistical analyses for glycan intensities relative to the internal standard Man6 from urine samples of 60 persons of which 13 were diagnosed for bladder cancer and 47 did not suffer from bladder cancer by Student's t-test. The intensities for the glycosphingolipid-derived glycans nLc4 and Gb3 are significantly (p≤0.05) increased in the group with bladder cancer compared to the control group. For the marker lactose, the significance threshold was slightly failed.
TABLE-US-00006 TABLE 3 Bladder cancer Control p value Marker n mean std. dev n mean std. dev t-test nLc4 13 7.60 13.72 47 2.27 4.92 0.0297 lactose 13 205.92 187.24 47 119.33 128.64 0.0578 Gb4 13 27.54 30.75 47 21.07 18.57 0.3443 Gb3 13 101.81 127.14 47 53.83 49.12 0.0391
Example 2: Detection of Bladder Cancer by Immunological Analysis of nLc4 in Urine Samples
[0059] An antibody against nLc4, e.g. a commercially available antibody or an antibody derived from hybridoma cells generated from animals that were experimentally immunized with nLc4 can be used to detect nLc4 in the urine sample of patients with bladder cancer applying e.g. an ELISA (enzyme linked immuno sorbent assay) or in a RIA (radio immuno assay) or by immunochromatography, e.g. by lateral flow test. The antibody can be polyclonal or monoclonal. The sample could be the glycosphingolipid fraction isolated from a urine sample, with the β-glycosidic bond of glycosphingolipids optionally hydrolysed to release the free glycans. Alternatively, the sample could be the urine sample without pre-treatment, or the sample could be the urine sample, complete or with cells removed, optionally containing an added detergent, further optionally treated with ultrasound, to release glycosphingolipids from membranes. Alternatively or additionally, the urine sample, complete or with cells removed, could be treated by addition of a glycanase, e.g. a ceramide glycanase, to release the glycans from membrane-bound, respectively from solubilized, glycosphingolipids.
Example 3: nLc4 for Use in the Immunotherapeutical Treatment Against Bladder Cancer
[0060] In this embodiment, immunotherapy includes nLc4 for use in the treatment of bladder cancer. Patients suffering from bladder cancer can be administered (vaccinated) with a glycan comprising or consisting of nLc4 and/or nLc4-ceramide and/or a nLc4-protein conjugate and/or a nLc4-derivative in order to provoke a T-cell dependent or innate immune response against nLc4 in these patients that is then directed against the bladder cancer expressing the antigen epitope nLc4. The immune response can be supported by vaccination in combination with a T-cell carrier (e.g. keyhole limpet hemocyanin) or with adjuvants (e.g. Mycobacterium cell-wall skeleton or QS-21 or saponin-based adjuvant).
Example 4: Immunotherapy Against Bladder Cancer by Using an Antibody Specific for nLc4
[0061] In this embodiment, an antibody that is specific for nLc4 is for use in the treatment of bladder cancer, which antibody is directed against nLc4 as a molecular target.
[0062] A monoclonal or polyclonal antibody against nLc4, e.g. a commercially available antibody or an antibody derived from hybridoma cells generated from animals that were experimentally immunized with nLc4 can be applied for immunotherapy against bladder cancer. Accordingly, the antibody specific for nLc4 can be a human or a non-human antibody.
[0063] For immunotherapy, patients can be directly administered with the nLc4-specific (monoclonal) antibody. This antibody can then activate complement- or cellular-dependent anticancer activities. Such an antibody might be also linked to a cytotoxic agent, e.g. the antibody can be biotinylated and then linked a (strept)avidin-tagged cytotoxic protein, e.g. saporin, a ribosome-inactivating toxin or directly linked to a cytotoxic agent, e.g. ricin chain A toxin or linked to a cytokine. Alternatively an anti-nLc4 antibody can be used to generate a monoclonal anti-idiotype antibody by immunization of experimental animals and generation of hybridoma cells. The anti-idiotype monoclonal antibody can be applied as an anti-idiotype vaccine as it mimics the nLc4 epitope and can elicit an anti-nLc4 antibody response that specifically reacts with tumour cells expressing nLc4.
Example 5: CAR Comprising Protein Binding of nLc4 for Use in the Treatment of Bladder Cancer
[0064] The paratope of an antibody binding to nLc4 is used to generate a respective single-chain variable fragment (scFv) binding to nLc4. This scFv was used as a portion of a CAR (chimeric antigen receptor), which from N-terminus to C-terminus contained the single-chain variable fragment antibody domain (scFv), a modified hCD8 hinge, a hCD8 transmembrane domain, an intracellular hCD28 signalling domain and an intracellular hCD3 (hCD3 zeta) signalling domain, which preferably are linked to one another from N-terminus to C-terminus, more preferably directly linked to one another form N-terminus to C-terminus. The CAR can contain a hΔFc IgG domain in the alternative to a modified hCD8 hinge, and/or a fusion hCD28 transmembrane domain—hCD28/CD3 zeta domain in the alternative to a hCD8 transmembrane domain, an intracellular hCD28 signalling domain and an intracellular hCD3 (hCD3 zeta) signalling domain.
[0065] A nucleic acid sequence encoding the CAR containing a scFv domain having specificity for nLc4 could be expressed in immune cells, e.g. T-cells, preferably in primary T-cells or NK cells, which cells could be introduced into an experimental animal having bladder cancer. The immune cells expressing the CAR reduced the presence of bladder cancer in the recipient.